Abstract
The methylation of erythrocyte membrane components in sickle cell anemia has been studied and found to differ considerably from that of normal erythrocytes. When sickle erythrocytes were incubated under physiological conditions (pH = 7.4, 37 degrees C) in the presence of L-[methyl-3H]methionine or S-adenosyl-L-[methyl-3H] methionine, a 50% decrease in the protein-carboxyl methylation was observed compared to the normal erythrocyte. This reduction in degree of methylation was reflected in all of the major methylated protein bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Since this methylation is catalyzed by the cytosolic protein methylase II (S-adenosylmethionine:protein-carboxyl O-methyltransferase, EC 2.1.1.24), the in vitro substrate capability of sickle erythrocyte ghosts and pH 11 treated ghosts were tested by incubation with purified protein methylase II and S-adenosyl-L-[methyl-14C]methionine. Both types of sickle cell ghost preparations showed the same 50% decrease in methylation as was seen in the intact cells. Since it was also shown that the protein methylase II and methyl acceptor membrane protein levels in the sickle erythrocytes are the same as the normal control, the data suggest that the observed reduced methylation may be due to an altered membrane conformation. The methylation of phospholipid was also studied and found to be decreased in sickle cell erythrocytes. However, this methylation was extremely minor in comparison to protein-carboxyl methylation which represents the bulk of the membrane methylation.
Highlights
When sickle erythrocytes were incubated under phys- have a profound effect on membrane structure and function iological conditions(pH = 7.4,37 “C) in the presence of ~-[methyZ-~H]methionineor S-adenosyl-~-[methyl’H]
Erythrocytes from sickle cell anemia are known to be demethionine,a 50% decreaseintheprotein-carboxyl methylation was observedcompared to thenormal erythrocyte
We report that the protein-carboxyl methylerythrocyte ghosts and pH11 treated ghosts were ation which represents the bulk of membrane methylation is tested by incubation with purified protein methyl1a1se markedly decreased
Summary
15 Ci/mmol) were purchased from Amersham methylationof phospholipidwas studied and found to be decreased in sickle cell erythrocytes This methylation was extremely minor in comparison to protein-carboxyl methylation which represents the Nuclear Corp. Methyl labeling of the cells was performed in a totalvolume of 1ml containing 0.5 ml of the protein-carboxyl methylation In bacteria, it has been shown washed erythrocytes, 100pmol of sodium phosphate, pH 7.4, and 0.14 that the specific methylation and demethylation of glutamyl m o l of [methyl-3H]AdoMet’(10pCi) or 1.87 nmol of ~-[methyl-’H]. Washed erythrocytes (0.5 ml) were incubated either with [rnethyl'HIAdoMet or L-[methyl-"Hlmethionine and membranes were prepared as described under"Experimental Procedures." Results are expressed P S mean k S.D. Protein methylaseI1activity was estimated as described elsewhere [17].One unit of enzyme activity is defined as 1pmol of methyl group transferred at 37 "C/min.
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